muramidase and aluminum-phosphate

Evaluation of the physical characteristics of vaccines formulated in the presence of adjuvants, such as aluminum salts (Alum), is an important step in the development of vaccines. Depending on the formulation conditions and the associated electrostatic interactions of the adjuvant particles, the vaccine suspension may transition between flocculated and deflocculated states. The impact of practical formulation parameters, including pH, ionic strength, and the presence of model antigens, has been correlated to the sedimentation behavior of aluminum phosphate suspensions. A novel approach for the characterization of suspension properties of Alum has been developed to predict the flocculated state of the system using a sedimentation analysis-based tool (Turbiscan®). Two sedimentation parameters, the settling onset time (Sonset) and the sedimentation volume ratio (SVR) can be determined simultaneously in a single measurement. The results demonstrate the suspension characteristics to be significantly altered by solution conditions (pH and ionic strength) and the charge state of bound antigens. Formulation conditions that promote the flocculated state of the suspension are characterized by faster Sonset and higher SVR, and are generally easy to resuspend. The Turbiscan® method described herein is a useful tool for the characterization of aluminum-containing suspensions and may be adapted for screening and optimization of suspension-based vaccine formulations in general.

Topics: Alum Compounds; Aluminum Compounds; Animals; Antigens; Cattle; Flocculation; Hydrogen-Ion Concentration; Muramidase; Osmolar Concentration; Particle Size; Phosphates; Serum Albumin, Bovine; Vaccine Potency; Vaccines

The degree of antigen adsorption by aluminum-containing adjuvants is considered an important characteristic of vaccines that is related to immunopotentiation by the adjuvant. This study examined immunopotentiation by aluminum phosphate adjuvant in three model vaccines in which the antigen was not adsorbed in the vaccine formulation nor when mixed in vitro with interstitial fluid. In the first model vaccine, aluminum phosphate adjuvant was pre-treated with 0.5 M KH2PO4 to minimize the adsorption of dephosphorylated alpha casein. The second model vaccine was composed of aluminum phosphate adjuvant and ovalbumin that was dephosphorylated by treatment with potato acid phosphatase. The third model vaccine consisted of aluminum phosphate adjuvant and lysozyme (LYS). In order to prevent adsorption of lysozyme, the aluminum phosphate adjuvant was pre-treated with fibrinogen, a protein present in interstitial fluid that binds strongly to aluminum phosphate adjuvant. Immunopotentiation was evaluated by measuring antibody production in mice. It was found that all three model vaccines induced antibody titers that were statistically higher than induced by a solution of antigen without adjuvant and similar to vaccines in which the antigens were adsorbed by aluminum phosphate adjuvant. Confocal microscopy experiments suggested that the antigens used in these experiments, even though not adsorbed to the aluminum phosphate adjuvant, were trapped in void spaces within the adjuvant aggregates, resulting in uptake of antigen by dendritic cells.

Topics: Adjuvants, Immunologic; Adsorption; Aluminum Compounds; Animals; Antibody Formation; Antigens; Caseins; Female; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Muramidase; Ovalbumin; Phosphates; Phosphorylation; Vaccines

The effect of the degree of adsorption of lysozyme by aluminium hydroxide adjuvant on the immune response in rabbits was studied. The surface charge of the adjuvant was modified by pretreatment with phosphate anion to produce five vaccines having degrees of adsorption ranging from 3 to 90%. The degree of adsorption of vaccines exhibiting 3, 35 or 85% adsorption changed to 40% within 1 h after each vaccine was mixed with sheep interstitial fluid to simulate subcutaneous administration. The mean anti-lysozyme antibody titers produced by the vaccines were the same and were four times greater than that produced by a lysozyme solution. Thus, the degree of adsorption of lysozyme in sheep interstitial fluid rather than the degree of adsorption in the vaccine correlated with the immune response.

Topics: Adjuvants, Immunologic; Adsorption; Aluminum Compounds; Aluminum Hydroxide; Animals; Antibody Formation; Extracellular Space; Immunization; Muramidase; Phosphates; Rabbits; Vaccines

The ability of interstitial fluid to change the degree of adsorption of ovalbumin to aluminum hydroxide adjuvant or lysozyme to aluminum phosphate adjuvant was studied. Ovalbumin and lysozyme were almost completely eluted after exposure at 37 degrees C to sheep lymph fluid for 4h or 15 min, respectively. The ability of sheep lymph fluid to elute lysozyme from aluminum phosphate adjuvant did not change as the model vaccine aged. However, only 60% of the ovalbumin adsorbed to aluminum hydroxide adjuvant was eluted during exposure to sheep lymph fluid for 24h after the model vaccine aged for 11 weeks at 4 degrees C.

Topics: Adjuvants, Immunologic; Adsorption; Aluminum Compounds; Aluminum Hydroxide; Animals; Drug Stability; Drug Storage; Egg Proteins; Lymph; Muramidase; Ovalbumin; Phosphates; Sheep

The effect of adsorbing two model proteins, bovine serum albumin and lysozyme, on the point of zero charge of aluminium-containing vaccine adjuvants was studied. At physiological pH, the adsorption of the negatively charged albumin (isoelectric point = 5.0) by aluminium hydroxide adjuvant (point of zero charge = 11.1) resulted in a decrease in the point of zero charge. In contrast, the adsorption of positively charged lysozyme (isoelectric point = 9.6) by the negatively charged aluminium phosphate adjuvant (point of zero charge = 5.0) resulted in an increase in the point of zero charge. The surface charge characteristics of the aluminium-containing adjuvant dominated at low protein coverage. In contrast, the surface charge characteristics of the adsorbed protein dominated at high protein coverage. Therefore, the physicochemical properties of the antigen-adjuvant complex and not the adjuvant alone should be considered during vaccine preparation.

Topics: Adjuvants, Immunologic; Adsorption; Aluminum Compounds; Aluminum Hydroxide; Animals; Cattle; Chemical Phenomena; Chemistry, Physical; Isoelectric Point; Muramidase; Phosphates; Serum Albumin, Bovine; Surface Properties