Change in the degree of adsorption of proteins by aluminum-containing adjuvants following exposure to interstitial fluid: freshly prepared and aged model 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.

Degree of antigen adsorption in the vaccine or interstitial fluid and its effect on the antibody response in rabbits.

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.

Detoxification of endotoxin by aluminum hydroxide adjuvant.

Langmuir adsorption isotherms of endotoxin and aluminum-containing adjuvants at pH 7.4 and 25 degrees C revealed that aluminum hydroxide adjuvant has a greater adsorption capacity (283 microg/mg Al) and adsorption coefficient (1.3x10(4) ml/miccrog) than aluminum phosphate adjuvant (3.0 microg/mg Al, 0.20 ml/microg). The difference in endotoxin adsorption was related to two adsorption mechanisms: electrostatic attraction and covalent bonding. The isoelectric point (iep) of endotoxin is approximately 2. An electrostatic attractive force will be present with aluminum hydroxide adjuvant (iep=11.4), and an electrostatic repulsive force will operate with aluminum phosphate adjuvant (iep=4.6). Endotoxin contains two phosphate groups in the lipid A portion. Covalent bonding occurs with surface aluminum in aluminum hydroxide adjuvant but is inhibited by surface phosphate in aluminum phosphate adjuvant. In-vitro desorption experiments using components of interstitial fluid showed that endotoxin adsorbed by aluminum hydroxide adjuvant was not desorbed by interstitial anions (5 mM phosphate or 2.7 mM citrate) or interstitial proteins (25 mg albumin/ml). The effect of aluminum-containing adjuvants on the systemic response of Sprague-Dawley rats to a 15 microg/kg subcutaneous dose of endotoxin was determined by measuring the serum concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). TNF-alpha and IL-6 were observed in the group which received an endotoxin solution or endotoxin and aluminum phosphate adjuvant. No TNF-alpha or IL-6 was detected in the group that received endotoxin and aluminum hydroxide adjuvant. Aluminum hydroxide adjuvant detoxifies endotoxin by adsorbing it in the vaccine and then not releasing it in interstitial fluid upon administration.

Aluminium phosphate adjuvants prepared by precipitation at constant pH. Part I: composition and structure.

Aluminium phosphate adjuvant was precipitated under constant pH conditions in an effort to characterize materials formed at defined precipitation conditions. A reaction vessel was designed to provide a continuous steady-state process. An aqueous solution containing aluminium chloride and sodium dihydrogen phosphate was pumped into the reaction vessel at a constant rate. A second pump infused a sodium hydroxide solution at the rate required to maintain the desired pH. Precipitations were performed between pH 3.0 and 7.5, at intervals of pH 0.5. The adjuvants were characterized using 27Al NMR, FTIR, Raman and X-ray diffraction methods along with elemental analysis. The results of this study indicate that a continuum of amorphous aluminium hydroxyphosphates were formed having properties that changed as a continuous function of the precipitation pH. The phosphate content decreased as the pH of precipitation increased. 27Al NMR spectra revealed that the majority of the aluminium was octahedrally coordinated, with a small percentage of tetrahedrally coordinated aluminium. The density of the adjuvants was directly related to the pH of precipitation. The most prominent feature of the IR and Raman spectra is the P-O stretching vibration of the structural PO(4) groups. The positions of these bands decreased linearly as the precipitation pH increased. The results of selective deuteration FTIR experiments are consistent with high surface area materials as most of the OH groups were exposed near the surface of the adjuvant.

Aluminium phosphate adjuvants prepared by precipitation at constant pH. Part II: physicochemical properties.

The impact of the pH of precipitation on the physicochemical properties of aluminium phosphate adjuvants was investigated by precipitating aluminium phosphate adjuvants under constant pH conditions at pH values from 3.0 to 7.5 at intervals of 0.5. The pH of precipitation did not affect the morphology, but the point of zero charge (PZC) and rate of acid neutralization varied directly with pH of precipitation. Aggregation and protein adsorption capacity exhibited a parabolic relationship to the pH of precipitation. Minimum protein adsorption and maximum aggregation were observed at pH 5.5. In contrast to adjuvants precipitated from the same reactants but under uncontrolled pH conditions, the pH of all of the adjuvants precipitated under constant pH conditions remained constant for a 3-month aging period at room temperature.

Stability of aluminium-containing adjuvants during aging at room temperature.

Aluminium phosphate adjuvant and aluminium hydroxide adjuvant became more ordered during aging at room temperature. The increased degree of order was accompanied by a decrease in protein adsorption capacity.

Analysis of aluminum hydroxyphosphate vaccine adjuvants by (27)Al MAS NMR.

The present study explores the use of (27)Al magic-angle-spinning (MAS) NMR for the characterization of aluminum hydroxyphosphate adjuvants. Adjuvants were prepared by two different methods: batch-precipitation and precipitation at constant pH, using a wide range of different conditions. The adjuvant compositions showed no evident stoichiometric restrictions and varied as a function of the precipitation conditions. All the aluminum hydroxyphosphate adjuvants were found by (27)Al MAS NMR to contain both tetrahedrally and octahedrally coordinated aluminum. The octahedral form was always predominant. The chemical shifts corresponding to octahedral aluminum were at values intermediate between that of aluminum hydroxide (9 ppm) and those of phosphate-containing aluminum minerals such as variscite (-9 ppm) and varied with the phosphate content of the adjuvant. This was true even for adjuvants precipitated above pH 6 indicating that the phosphate is incorporated into the bulk solid phase contrary to predictions in the literature. Aside from the presence of tetrahedral and octahedral aluminum, there was no evidence in any of the adjuvants of distinct, structurally defined phases indicating that the adjuvants are not mixtures of distinct phases which differ significantly in the number of phosphorus atoms in the next-nearest-neighbor (NNN) position to aluminum.

The in vitro displacement of adsorbed model antigens from aluminium-containing adjuvants by interstitial proteins.

Vaccines prepared by adsorbing an antigen onto an aluminium-containing adjuvant are usually administered by intramuscular or subcutaneous injection. The vaccine then comes in contact with interstitial fluid which contains proteins. In vitro displacement studies were performed to determine whether antigens, which are adsorbed to aluminium-containing adjuvants, can be displaced by interstitial proteins. It was found that when previously adsorbed model antigens such as lysozyme or myoglobin were exposed to interstitial proteins such as albumin or fibrinogen that extensive displacement occurred. A factorial study of the displacement of myoglobin from aluminium hydroxide adjuvant by albumin was performed. The displacement occurred rapidly with the majority of the displacement occurring in less than 15 min. Whether the concentration of the adsorbed myoglobin was above or below the adsorptive capacity of the aluminium hydroxide adjuvant affected the amount which could be displaced. Less myoglobin was displaced when the concentration was below the adsorptive capacity. The age of the model vaccine (1, 2 or 7 days) prior to exposure to the interstitial protein did not influence the amount of myoglobin that was displaced. The affinity of model antigens and interstitial proteins for aluminium hydroxide or aluminium phosphate adjuvant was characterized by the adsorption coefficient in the Langmuir equation. In every case studied, the protein having the larger adsorption coefficient was able to displace the protein with the smaller adsorption coefficient.

Stability of aluminium-containing adjuvants to autoclaving.

Aluminium phosphate adjuvant remained amorphous when autoclaved for 30 or 60 min at 121 degrees C. However, deprotonation and dehydration reactions occurred as evidenced by a decrease in the pH. The protein adsorption capacity, rate of acid neutralization at pH 2.5 and point of zero charge also decreased indicating that the deprotonation/dehydration reactions resulted in a decreased surface area. Autoclaving aluminium hydroxide adjuvant increased the degree of crystallinity as measured by the width at half height of the major band in the X-ray diffractogram. The pH decreased during autoclaving suggesting that the same deprotonation/dehydration reactions which reduced the surface area of aluminium phosphate adjuvant were responsible for the increased degree of crystallinity. These reactions also resulted in a reduced surface area as both the protein adsorption capacity and viscosity decreased following autoclaving.

In vivo absorption of aluminium-containing vaccine adjuvants using 26Al.

Aluminium hydroxide (AH) and aluminium phosphate (AP) adjuvants, labelled with 26Al, were injected intramuscularly (i.m.) in New Zealand White rabbits. Blood and urine samples were collected for 28 days and analysed for 26Al using accelerator mass spectrometry to determine the absorption and elimination of AH and AP adjuvants. 26Al was present in the first blood sample (1 h) for both adjuvants. The area under the blood level curve for 28 days indicates that three times more aluminium was absorbed from AP adjuvant than AH adjuvant. The distribution profile of aluminium to tissues was the same for both adjuvants (kidney > spleen > liver > heart > lymph node > brain). This study has demonstrated that in vivo mechanisms are available to eliminate aluminium-containing adjuvants after i.m. administration. In addition, the pharmacokinetic profiles of AH and AP adjuvants are different.

Role of the electrostatic attractive force in the adsorption of proteins by aluminum hydroxide adjuvant.

The fact that both aluminum hydroxide adjuvant and proteins have a pH dependent surface charge means that electrostatic forces play a role in the adsorption of proteins by aluminum hydroxide adjuvant during the preparation of vaccines. The objective of this study was to examine the contribution of the electrostatic attractive force in the adsorption of proteins by aluminum hydroxide adjuvant. Since the surface charge characteristics of aluminum hydroxide adjuvant can be modified by the adsorption of phosphate anion, a series of aluminum hydroxide adjuvants were prepared by treatment with various concentrations of phosphate anion. The isoelectric points (iep) of these adjuvants ranged from 11.0 to 4.6 and the electrophoretic mobilities at pH 7.4 ranged from 2.0 to -3.3 microns cm/V s. The line broadening of the (020) band of the X-ray diffraction pattern indicated that treatment with phosphate anion did not change the primary crystallite dimension. Adsorption at pH 7.4 of positively charged lysozyme (iep = 11.1) was directly related to the negative surface charge of the adjuvant. No adsorption occurred when the surface charge was positive. In contrast, negatively charged ovalbumin (iep = 4.6) was adsorbed by all of the adjuvants at pH 7.4, although the adsorptive capacity was the greatest when the surface charge was positive. The results indicate that adsorptive forces in addition to the electrostatic attractive force play an important role in the adsorption of some proteins by aluminum hydroxide adjuvant. It is believed the structurally flexible proteins, like ovalbumin, exhibit more complex adsorption behavior than structurally rigid proteins, like lysozyme, for which adsorptive behavior can be explained by electrostatic forces.

Treatment of aluminium hydroxide adjuvant to optimize the adsorption of basic proteins.

Aluminium hydroxide adjuvant has an isoelectric point (i.e.p.) of ca 11 and is a good adsorbent for acidic proteins due to the contribution of electrostatic attractive forces. However, electrostatic repulsive forces reduce its ability to adsorb basic proteins. Pretreatment of aluminium hydroxide adjuvant with carefully selected concentrations of phosphate anion reduces the positive surface charge which exists at pH 7.4. Treatment with higher concentrations of phosphate anion produces a negative surface charge. The adsorption of lysozyme (i.e.p = 11.1) was found to be directly related to the concentration of phosphate anion used to pretreat the aluminium hydroxide adjuvant.

Structure and properties of aluminum-containing adjuvants.

This chapter is concerned with the identification, characterization, and behavior of aluminum-containing adjuvants with proteins and anions similar to those occurring in vaccines and interstitial fluid. Aluminum-containing adjuvants referred to commercially as aluminum hydroxide have been identified as poorly crystalline aluminum oxyhydroxide with the structure of the mineral boehmite. Relevant properties of this material include its high surface area and its high pI, which provide the adjuvant with a high adsorptive capacity for positively charged proteins. Aluminum phosphate and alum-precipitated adjuvants may be classified as amorphous aluminum hydroxyphosphate with little or no specifically adsorbed sulfate. Variations in the molar PO4/A1 ratio of amorphous aluminum hydroxyphosphates result in PI values that range from 5 up to 7; the materials are negatively charged at a physiological pH of 7.4. The amorphous nature of these compounds gives them high surface area and high protein adsorptive capacity for positively charged proteins. Observations on the interactions of anions and charged proteins with charged adjuvant surfaces have provided a framework for predicting behavior of complex systems of vaccines and for designing specific combinations of adjuvants and antigens to optimize the stability and efficacy of vaccines.

Effect of protein adsorption on the surface charge characteristics of aluminium-containing adjuvants.

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.

Relationship between protein adsorptive capacity and the X-ray diffraction pattern of aluminium hydroxide adjuvants.

Thermal treatment during the preparation of aluminium hydroxide adjuvants affects the primary crystallite size of the adjuvant. The primary crystallite size can be characterized by the line broadening of the (020) reflection of the X-ray diffraction pattern. Studies of protein adsorption using bovine serum albumin as a model protein revealed a direct relationship between the albumin adsorptive capacity and the width at half height (WHH) of the (020) reflection in the X-ray diffraction pattern.

The importance of surface charge in the optimization of antigen-adjuvant interactions.

The adsorptive behavior of the recombinant malarial antigens R32tet32, R32NS181 and NS181V20 to aluminum hydroxide and aluminum phosphate gels was studied as a function of pH and buffer ions. The Plasmodium falciparum antigen, R32NS181, and the P. vivax antigen, NS181V20, with isoelectric points (pI) of 5.9 and 5.5, respectively, adsorbed readily to the positively charged boehmite form of aluminum hydroxide gel. These two antigens displayed reversible, linear adsorption behavior in the pH range 5-9, with maximal adsorption observed at the lowest pH studied. The addition of acetate buffer ions had little effect on adsorption, while the presence of phosphate decreased adsorption for R32NS181 and NS181V20 by 25 and 40% respectively. The adsorptive behavior of these two antigens with the negatively charged adjuvant, aluminum phosphate, was markedly decreased. The converse situation was observed with the R32tet32 antigen, whose pI is estimated to be 12.8. There was minimal interaction of this antigen with aluminum hydroxide gel except in the presence of phosphate counter ions and significant, nonreversible adsorption with aluminum phosphate gel. Enhanced adsorption of R32tet32 to aluminum hydroxide gel in the presence of phosphate is suggested to be the result of a covalent bond between a surface aluminum and a phosphate anion that modifies the surface charge of the aluminum hydroxide gel. These results indicate that the role of complementary surface charges, both for the ionization state of the protein and for the aluminum adjuvants, is the key in optimizing conditions for significant antigen-adjuvant interactions.

Predicting the adsorption of proteins by aluminium-containing adjuvants.

The adsorption of two model proteins, albumin and lysozyme, by boehmite or amorphous aluminium hydroxyphosphate adjuvants was studied. Electrostatic, attraction has a major role in adsorption. At physiological pH, boehmite, which has a point of zero charge above 7.35, extensively adsorbed albumin, which has an isoelectric point of 4.8, but was not effective in adsorbing lysozyme (isoelectric point, 11.0). Conversely, amorphous aluminium hydroxyphosphate (point of zero charge, 4.0) was effective in adsorbing lysozyme but adsorbed relatively little albumin. The results suggest that the selection of either boehmite or amorphous aluminium hydroxyphosphate as an adjuvant should be based in part on the isoelectric point of the antigen.

Adsorption of phosphate by aluminum hydroxycarbonate.

Phosphate is specifically adsorbed by aluminum hydroxycarbonate by anion ligand exchange. IR analysis indicated that phosphate exchanged with specifically adsorbed carbonate. Adsorption is favored by low pH and is inversely related to particle size. Adsorption of phosphate decreases the rate of acid neutralization of aluminum hydroxycarbonate. The results are applied to the treatment of hyperphosphatemia and hypophosphatemia.

Potential of organic cation-saturated montmorillonite as treatment for poisoning by weak bases.

The preferential adsorption of the protonated form of weak bases by montmorillonite causes an increase in the pKeff of atrazine. The effect on the acid-base equilibrium of atrazine is related to the exchangeable cation on the surface of montmorillonite. The greatest effect was produced by the presence of 3-hydroxypropylammonium-saturated montmorillonite, which caused the pKeff of atrazine to increase 5.3 units to 6.9. This shift in pKeff causes the protonated form of atrazine to be the predominate species in the pH range encountered in the GI tract and should result in a high degree of adsorption of atrazine. Fraction-bound studies confirmed this hypothesis by demonstrating virtually complete absorption of atrazine by 3-hydroxypropylammonium-saturated montmorillonite up to pH 6. The fraction-bound studies also verified that protonated atrazine is adsorbed more completely by 3-hydroxypropylammonium-saturated montmorillonite than by bentonite USP or sodium-saturated montmorillonite. It is believed that saturation of the clay surface by an organic cation alters the surface environment of the clay, which results in enhanced adsorption of the protonated form of atrazine. The potential utility of montmorillonite saturated with an organic cation as an adsorbent for the emergency treatment of poisoning by weak bases is suggested.