Secondary structure analysis of HIV-1-gp41 in solution and adsorbed to aluminum hydroxide by Fourier transform infrared spectroscopy.

The formulation of human vaccines often includes adjuvants such as aluminum hydroxide that are added to enhance the immune responses to vaccine antigens. However, these adjuvants may also affect the conformation of antigenic proteins. Such structural modifications could lead to changes in antigenicity such that suboptimal protective immune responses could be generated relative to those induced by the vaccine antigens alone. Here, we used attenuated total reflectance infrared spectroscopy (ATR-FTIR) to compare the secondary structures of recombinant HIV-1-gp41 (gp41) in solution or adsorbed to aluminum hydroxide. The gp41 secondary structure content was 72% alpha-helices and 28% beta-sheets in 5 mM formate buffer p(2)H 2.5, while it was 66% beta-sheets and 34% random coil in acetonitril/(2)H(2)O (95/5:v/v). A fully reversible conformational change of gp41 in acetonitril/(2)H(2)O (95/5:v/v) was observed upon addition of either 35 mM formate p(2)H 2.5 or 0.1% (w/v) detergent (Tween 20, Hecameg, Brij 35 or beta-d-octyl-glucopyranoside). When gp41 was adsorbed to aluminum hydroxide in the presence of 0.1% (w/v) detergent, in either formate or in acetonitril/(2)H(2)O (95/5:v/v) its secondary structure remained stable and was identical to that of gp41 in 5 mM formate buffer p(2)H 2.5. The method described here could be applied for the characterization of gp41 conformers for use in immunological screening of antigens, and more generally to all antigenic proteins adsorbed to aluminum hydroxide.

Aluminium assay and evaluation of the local reaction at several time points after intramuscular administration of aluminium containing vaccines in the Cynomolgus monkey.

Aluminium hydroxide and aluminium phosphate have been widely used as vaccine adjuvants with a good safety record for several decades. The recent observation in human deltoid muscle of macrophage aggregates containing aluminium hydroxide spicules and termed Macrophagic Myofasciitis (MMF) has encouraged research on aluminium salts. This study was conducted in order to further investigate the clearance of aluminium at the vaccine injection site and the features of induced histopathological lesions. Two groups of 12 monkeys were immunised in the quadriceps muscle with Diphtheria-Tetanus vaccines, which were adjuvanted with either aluminium hydroxide or aluminium phosphate. Three, six or twelve months after vaccination, four monkeys from each group were sacrificed and histopathological examination and aluminium assays were performed on quadriceps muscle sections. Histopathological lesions, similar to the MMF described in humans, were observed and were still present 3 months after aluminium phosphate and 12 months after aluminium hydroxide adjuvanted vaccine administration. An increase in aluminium concentration, more marked in the area of the lesions, was also observed at the 3- and 6-month time points. These findings were localised at the injection site and no similar changes were observed in the distal or proximal muscle fragments. We conclude from this study that aluminium adjuvanted vaccines administered by the intramuscular route trigger histopathological changes restricted to the area around the injection site which persist for several months but are not associated with abnormal clinical signs.

Aluminum hydroxide adjuvant induces macrophage differentiation towards a specialized antigen-presenting cell type.

Aluminum hydroxide (AlOOH) has been used for many years as a vaccine adjuvant, but little is known about its mechanism of action. We investigated in this study the in vitro effect of aluminum hydroxide adjuvant on isolated macrophages. We showed that AlOOH-stimulated macrophages contain large and persistent intracellular crystalline inclusions, a characteristic property of muscle infiltrated macrophages described in animal models of vaccine injection, as well as in the recently described macrophagic myofasciitis (MMF) histological reaction in humans. AlOOH-loaded macrophages exhibited phenotypical and functional modifications, as they expressed the classical markers of myeloid dendritic cells (HLA-DR(high)/CD86(high)/CD83(+)/CD1a(-)/CD14(-)) and displayed potent ability to induce MHC-II-restricted antigen specific memory responses, but kept a macrophage morphology. This suggests a key role of macrophages, in the reaction to AlOOH-adjuvanted vaccines and these mature antigen-presenting macrophages may therefore be of particular importance in the establishment of memory responses and in vaccination mechanisms leading to long-lasting protection.